Project Summary Production of HIV-1 envelope protein [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] for clinical studies is a critical objective for vaccine development. Its conformational plasticity, which allows it to adopt multiple conformational states important for function, often makes its expression and purification very difficult. Our recent work shows a route for overcoming this problem. First, we have identified two full-length gp160s from difficult-to-neutralize HIV-1 primary isolates that are conformationally homogenous when expressed on the surface of human cells. Their antigenic properties correlate closely with antibody neutralization. Second, truncation of the gp41 cytoplasmic tail (CT) has minimal impact on the activity of Env as a fusogen, but has an unexpected role in constraining the conformational variability of the ectodomain. Third, we have determined an atomic structure of the HIV-1 Env transmembrane domain (TMD), which is a well-ordered, ?-helical trimer. Introduction into Env of a set of mutations that disrupts the TMD trimer alters antibody sensitivity. Thus, both the TMD and the CT contribute to Env stability and antigenicity. In Phase I of this project, we will build on the three findings to identify specific structural determinants of HIV-1 Env stability and expression, consistent with its native antigenic properties. We will combine these structural determinants, along with structure-based modifications in the TMD, to develop a general strategy for high-yield production in mammalian cells of essentially any HIV-1 Env, regardless of isolate or clade. We will also explore various expression vectors and cell lines, to establish a robust production pipeline. In a projected Phase II, we will develop a purification scheme suitable for producing clinical-grade materials. We will also evaluate immunogenicity of those Envs in animal models. The Phase I Specific Aims include: 1) to identify structural determinants in HIV-1 Env that have significant impact on its stability and expression; 2) to develop a general strategy for mammalian-cell expression of full-length, HIV-1 Env trimers with native antigenic properties; 3) optimize expression conditions to maximize the yield of full-length, native HIV-1 Env. By the end of Phase I of this proposal, we expect to establish an expression system with high yield (>10-fold increase in protein levels over current well-expressed variants) of full-length HIV-1 Env trimers derived from many HIV-1 isolates. Such a system will provide a solid foundation for future Phase II studies to produce HIV-1 Env trimer immunogens suitable for clinical trials.